The ultimate goal of this project is to explore cellular and molecular replacement strategies for treating motor neuron degeneration. Our approach entails comprehensive analyses of both intrinsic properties and extrinsic factors that affect the survival of human motor neurons in adult almyotrophic lateral sclerosis (ALS) spinal cord. Our logic is based on the following premises: (1) Motor neuron degeneration in ALS may not be cell autonomous to motor neurons but may arise, in part, from an abnormal glial environment. Thus, modifying the glial environment will protect endogenous motor neurons and create a healthy milieu for grafted motor neurons. (2) We have established a system to efficiently produce healthy human glial cells, such as astrocytes, as well as motor neurons from naive embryonic stem cells (ESCs). These neural cells may represent a source of therapeutic agents, as well as a tool for dissecting neural cell interactions during motor neuron degeneration. (3) Human stem cells, including ESCs and neural stem/progenitor cells, can be genetically modified to carry therapeutic genes. This project will take advantage of advances in our understanding of intermittent hypoxia-induced respiratory motor neuron plasticity under investigation in Project 1, and build upon the foundation of motor neuron-astrocyte interactions in ALS pathogenesis assessed in Project 2. We will genetically modify hESCs (NIH Registry, WA01, WA09) to carry cell death-resistant genes and transplant the differentiated human motor neurons into the spinal cord of ALS rats. Through interactions with projects 1 & 2, we will evaluate whether and how intermittent hypoxia, a respiratory exercise, and growth factor-producing astrocytes promote the survival, integration, and axonal growth of the grafted human motor neurons. Our goal is to identify an ideal combinatory strategy to achieve successful transplantation of human motor neurons in the adult ALS spinal cord. Together with projects 1 & 2, we hope to arrive at a comprehensive strategy for treating the devastating ALS.